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Biomedical subjects

A M Miles

Publications and source records attributed to A M Miles.

At least 37 records · Page 2Linked to original sources
PubMed1996

Modulation of superoxide-dependent oxidation and hydroxylation reactions by nitric oxide.

The rapid and spontaneous interaction between superoxide (O2-.) and nitric oxide (NO) to yield the potent oxidants peroxynitrite (ONOO-) and peroxynitrous acid (ONOOH), has been suggested to represent an important pathway by which tissue may be injured during inflammation. Although several groups of investigators have demonstrated substantial oxidizing and cytotoxic activities of chemically synthesized ONOO-, there has been little information available quantifying the interaction between O2-. and NO in the absence or the presence of redox-active iron. Using the hypoxanthine (HX)/xanthine oxidase system to generate various fluxes of O2-. and H2O2 and the spontaneous decomposition of the spermine/NO adduct to produce various fluxes of NO, we found that in the absence of redox-active iron, the simultaneous production of equimolar fluxes of O2-. and NO increased the oxidation of dihydrorhodamine (DHR) from normally undetectable levels to approximately 15 microM, suggesting the formation of a potent oxidant. Superoxide dismutase, but not catalase, inhibited this oxidative reaction, suggesting that O2-. and not hydrogen peroxide (H2O2) interacts with NO to generate a potent oxidizing agent. Excess production of either radical virtually eliminated the oxidation of DHR. In the presence of 5 microM Fe+3-EDTA to insure optimum O2-.-driven Fenton chemistry, NO enhanced modestly HX/xanthine oxidase-induced oxidation of DHR. As expected, both superoxide dismutase and catalase inhibited this Fe-catalyzed oxidation reaction. Excess NO production with respect to O2-. flux produced only modest inhibition (33%) of DHR oxidation. In a separate series of studies, we found that equimolar fluxes of O2-. and NO in the absence of iron only modestly enhanced hydroxylation of benzoic acid from undetectable levels to 0.6 microM 2-hydroxybenzoate. In the presence of 5 microM Fe+3-EDTA, HX/xanthine oxidase-mediated hydroxylation of benzoic acid increased dramatically from undetectable levels to 4.5 microM of the hydroxylated product. Superoxide dismutase and catalase were both effective at inhibiting this classic O2-.-driven Fenton reaction. Interestingly, NO inhibited this iron-catalyzed hydroxylation reaction in a concentration-dependent manner such that fluxes of NO approximating those of O2-. and H2O2 virtually abolished the hydroxylation of benzoic acid. We conclude that in the absence of iron, equimolar fluxes of NO and O2-. interact to yield potent oxidants such as ONOO-/ONOOH, which oxidize organic compounds. Excess production of either radical remarkably inhibits these oxidative reactions. In the presence of low molecular weight redox-active iron complexes, NO may enhance or inhibit O2-.-dependent oxidation and hydroxylation reactions depending upon their relative fluxes.

Benzoates↗
PubMed1996

Hemodialysis immediately after acute myocardial infarction.

Acute myocardial infarction (AMI) is common in patients who have end-stage renal disease. However, the prudent interval after AMI until resuming hemodialysis is unknown. Also incidence and severity of intradialytic morbid events during the initial dialysis treatment after AMI have not been determined. We conducted a retrospective analysis of the course of hemodialyses performed immediately after AMI in 13 maintenance hemodialysis patients (group 1) hospitalized with AMI over the 5-year period 1988-1992. For comparison, the incidence of intradialytic morbid events (hypotension--systolic blood pressure < 90 or diastolic blood pressure < 60 mm Hg or a fall in systolic or diastolic blood pressure of > 30 mm Hg--with and without symptoms, arrhythmias, and unplanned termination of hemodialysis was extracted from the charts of 9 maintenance hemodialysis patients (group 2) admitted during the same period with angina but no AMI, and in 13 stable ambulatory hemodialysis patients (group 3) dialyzed during the same period who had no evidence of heart disease. Patients in groups 1 and 2 were sorted by time interval from onset of chest pain to initiation of hemodialysis (< 12, 12-24, and > 24 h). In group 1, we examined the relationship of anatomic location of AMI, number of antihypertensive medications, predialysis left ventricular systolic ejection fraction, and various other clinical and laboratory parameters to the incidence intradialytic morbid events. The mean (+/- SD) age of the study subjects was 67 +/- 7.5 years in group 1, 57 +/- 3.7 in group 2, and 60 +/- 11 years in group 3 (p = 0.6). Arrhythmias and early termination of dialysis did not occur in any patient. Intradialytic hypotension (IDH) was recorded in 5 (38%) of 13 patients in group 1, in 3 (33%) of 9 in group 2, and in 2 (15%) of 13 patients in group 3 (p = 0.47). 4 (80%) of 5 patients in group 1 had multiple episodes of IDH. There were 0.92 +/- 1.4 episodes of IDH per patient in group 1 as compared with a rate of 0.44 +/- 0.68 per patient in group 2, and of 0.15 +/- 0.36 per patient in group 3 (p = 0.2). IDH responded to 0.9% normal saline replacement in all cases. Group 1 patients who had IDH (n = 5) were older (68 +/- 3 vs. 58 +/- 7 years, p = 0.01), had a lower diastolic blood pressure at the start of hemodialysis (59 +/- 13 vs. 83 +/- 13 mm Hg; p = 0.01), had a lower post-AMI left ventricular systolic ejection fraction (42 +/- 19 vs. 62 +/- 10%; p = 0.04), and also had a lower predialysis serum albumin level (3.6 +/- 0.4 vs. 4.1 +/- 0.4 g/dl; p = 0.09) than those who did not have IDH (n = 8). All 5 group 1 patients who had IDH (100%) had had prior AMI as compared with 2 (25%) of 8 of those who did not have IDH (p = 0.02). AMI involved the inferior myocardial wall in more (4 of 5; 80%) of the group 1 patients who had IDH as compared with those who did not have IDH (2 of 8; 25%; odds ratio = 9.5; p = 0.08; 95% confidence interval = 0.7-341.0). In group 1 patients, the time from onset of chest pain to hemodialysis did not affect the risk of IDH (p = 0.4). We conclude that a low diastolic blood pressure at onset of hemodialysis prior myocardial infarction, inferior myocardial wall involvement, advanced age, and a low predialysis serum albumin level are risk factors for the development of hypotension during the first hemodialysis session after AMI.

Aged↗
PubMed1995

Photoinduced structural changes in the collagen/gelatin binding domain of fibronectin.

Prolonged exposure of human plasma fibronectin (pFn) and its 40- and 21-kDa collagen/gelatin binding fragments (GBFs) to 280-nm irradiation decreased their affinity for gelatin and for TR-CB7, a fluorescently labeled CNBr fragment of the alpha-1 chain of type I collagen. Fluorescence polarization binding assays of TR-CB7 with pFn and the 40-kDa GBF yielded progressively higher Kd's with increased time of exposure to 280-nm light at 25 degrees C. Binding of nonirradiated and irradiated pFn and fragments to gelatin-Sepharose correlated with the polarization data, confirming diminished gelatin binding following exposure to 280-nm light. Fluorescence spectra of intrinsic tryptophans in the 21- and 40-kDa GBFs exhibited changes indicative of photoinduced conformational changes; the maximum fluorescence wavelength red-shifted from between 340 and 350 nm to 360 nm, with concomitant increases in fluorescence intensity. Exposure of 21- and 40-kDa GBFs and pFn to 280-nm light also generated approximately two, four, and six free sulfhydryl groups per molecule, respectively. No sulfhydryl release was observed in other Trp- and disulfide-containing proteins under the same conditions. We propose that the fluorescence changes as well as the changes in affinity for gelatin or the collagen fragment result from structural changes secondary to the breakage of disulfide bonds, as a consequence of energy transfer from nearby tryptophans in one or more of the Fn type I repeats in the gelatin binding region of fibronectin.

Collagen↗
PubMed1995

Nitric oxide synthase in circulating vs. extravasated polymorphonuclear leukocytes.

It is becoming increasingly apparent that certain forms of acute and chronic inflammation are associated with enhanced production of nitric oxide (NO). Although substantial information has been obtained describing the regulation of NO synthase (NOS) in macrophages, little information is available regarding the biochemistry and molecular biology of NOS in circulating vs. extravasated polymorphonuclear leukocytes (PMNs). The objective of this study was to characterize the molecular and biochemical properties of the inducible NO synthase (iNOS) in circulating vs. extravasated rat and human PMNs. Circulating rat and human PMNs were purified from peripheral blood and extravasated PMNs were elicited in rats by intraperitoneal injection of 1% oyster glycogen or in humans by peritoneal dialysis of patients with peritonitis. Inducible NOS mRNA from circulating and elicited PMNs was quantified using slot blot hybridization analysis with a cDNA probe specific for iNOS. iNOS protein was identified using Western immunoblot analysis, and NOS activity was quantified by measuring the NG-monomethyl-L-arginine (L-NMMA)-inhibitable conversion of 14C-labeled L-arginine to L-[14C]citrulline. In a separate series of experiments, circulating or extravasated PMNs were cultured for 4 h and the accumulation of L-NMMA-inhibitable nitrite (NO2-) in the supernatant was determined and used as a measure of NO production in vitro. We found that circulating PMNs (rat or human) contained no iNOS mRNA, protein, or enzymatic activity. Furthermore, circulating rat or human PMNs (2 x 10(6) cells/well) were unable to generate significant amounts of NO2- when cultured for 4 h in vitro. In contrast, iNOS mRNA levels in 4- and 6-h elicited rat PMNs increased 21- and 42-fold, respectively, when compared with circulating cells. Western blot analysis revealed the presence of iNOS protein in the elicited rat PMNs and iNOS enzymatic activity increased from normally undetectable levels in circulating rat PMNs to 81 and 285 pmol/min/mg for the 4- and 6-h elicited rat PMNs, respectively. Approximately 20-30% of the total iNOS activity was Ca(2+)-dependent. Nitrite formation by elicited rat PMNs in the absence of any exogenous stimuli increased from normally undetectable amounts for circulating PMNs to approximately 8 and 11 microM/10(6) cells for the 4- and 6-h elicited PMNs, respectively. Highly enriched preparations of extravasated human PMNs contained neither message, protein nor iNOS enzymatic activity. Taken together our data demonstrate that inflammation-induced extravasation of rat PMNs upregulates the transcription and translation of iNOS in a time-dependent fashion and that 20-30% of the total inducible NOS is Ca(2+)-dependent. In contrast, neither circulating nor extravasated human PMNs contained iNOS message, protein, or enzymatic activity. These data suggest that the human PMN iNOS gene is under very different regulation than is the rat gene.

Animals↗
PubMed1995

Aspergillus peritonitis: therapy, survival, and return to peritoneal dialysis.

Peritonitis caused by fungi of the species Aspergillus is rare in peritoneal dialysis patients and is associated with high mortality. Four of the six reported survivors have been unable to resume peritoneal dialysis. We report the successful treatment of Aspergillus niger peritonitis with a combination of intravenous amphotericin B and peritoneal catheter removal in a patient on continuous ambulatory peritoneal dialysis (CAPD), and review previously reported cases of Aspergillus peritonitis. Our patient returned to CAPD after 2 months and has maintained good peritoneal transport 1 year after resumption of CAPD. The early recognition and aggressive treatment of peritonitis due to Aspergillus sp may allow eventual return to and successful maintenance of CAPD.

Amphotericin B↗
PubMed1995

Effects of superoxide on nitric oxide-dependent N-nitrosation reactions.

Recent studies have demonstrated that nitric oxide (NO) in the presence of superoxide (O2-) may mediate mutagenesis via the N-nitrosation of DNA bases followed by nitrosative deamination to yield their hydroxylated derivatives. We have found that phorbol myristate acetate (PMA)-activated extravasated rat neutrophils (PMNs) will N-nitrosate 2,3-diaminonaphthalene (DAN) to yield its highly fluorescent nitrosation product 2,3-naphthotriazole (triazole) via the L-arginine dependent formation of NO. Addition of SOD enhanced triazole formation suggesting that O2- production may inhibit the N-nitrosating activity and thus the mutagenic activity of inflammatory PMNs. The objective of this study was to assess the role of superoxide as a modulator of NO-dependent N-nitrosation reactions using PMA-activated PMNs as well as a chemically defined-system that generates both NO and superoxide. We found that PMA-activation of PMNs reduced found that PMA-activation of PMNs reduced the amount of N-nitrosation of DAN by approximately 64% when compared to non-stimulated cells (450 vs. 1250 nM). Addition of SOD but not inactivated SOD or catalase to PMA-activated PMNs enhanced the formation of triazole by approximately 4-fold (1950 nM). In addition, we found that the NO-releasing spermine/NO adduct (Sp/NO; 50 microM) which produces approximately 1.0 nmol NO/min generated approximately 8000 nM of triazole whereas the combination of Sp/NO and a superoxide generator (hypoxanthine/xanthine oxidase) that produces approximately 1.0 nmol O2-/min reduced triazole formation by 90% (790 nM). Addition of SOD but not catalase restored the N-nitrosating activity. We conclude that equimolar fluxes of superoxide react rapidly with NO to generate products that have only limited ability to N-nitrosate aromatic amino compounds and thus may have limited ability to promote mutagenesis via the nitrosative deamination of DNA bases.

2-Naphthylamine↗
PubMed1994

Effects of aminosalicylates and immunosuppressive agents on nitric oxide-dependent N-nitrosation reactions.

Recent studies have demonstrated that nitric oxide (NO) rapidly and spontaneously decomposes in oxygenated solutions to generate potent N-nitrosating agents. These electrophilic substances have been shown to mediate mutagenesis and carcinogenesis via the formation of aliphatic and aromatic nitrosamines. We have also demonstrated that extravasated neutrophils and macrophages produce significant amounts of N-nitrosating agents derived exclusively from NO. During the course of these studies, we found that certain antioxidants, including 5-aminosalicylic acid (5-ASA), inhibited the leukocyte-mediated N-nitrosation reaction. Because 5-ASA and other anti-inflammatory and immunosuppressive drugs are used to treat inflammatory bowel disease, we wondered if any of these other compounds might also modulate N-nitrosation reactions in vitro. Therefore, the objectives of this study were to assess the ability of aminosalicylates and certain immunosuppressive agents to inhibit NO-dependent N-nitrosation of a model aromatic amine (2,3-diaminonaphthalene) and to determine whether this inhibitory activity correlated with their oxidation potential. We found that the concentrations necessary to inhibit the N-nitrosation reaction by 50% (IC50) were 25, 50 and 100 microM for 5-ASA, olsalazine (dimeric 5-ASA) and sulfasalazine, respectively. In contrast, sulfapyridine, 4-ASA, N-acetyl-5-ASA, 6-mercaptopurine, azathioprine, and methotrexate were either much less effective or inactive at inhibiting the N-nitrosation reaction. Although 5-ASA was able to fully scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl, neither olsalazine nor sulfasalazine was found to be effective at scavenging this weak oxidant. We did find that olsalazine possessed an oxidation potential substantially less than that of sulfasalazine, suggesting that it may, in fact, scavenge more potent oxidizing agents such as the N-nitrosating agent. We conclude that 5-ASA and olsalazine inhibit NO-dependent N-nitrosation reactions by scavenging or decomposing the nitrosating agent(s). We propose that the secondary nitrogen unique to sulfasalazine interacts with the nitrosating agent to yield a secondary nitrosamine, thereby competing for N-nitrosation of our detector.

2-Naphthylamine↗
PubMed1993

Functional methionines in the collagen/gelatin binding domain of plasma fibronectin: effects of chemical modification by chloramine T.

Chemical modification of plasma fibronectin (pFn) or its 40-kDa collagen/gelatin binding (CGB) domain by low concentrations of chloramine T (CT), a methionine-specific oxidant, caused decreased binding affinity between pFn or the isolated CGB domain and Sepharose-immobilized denatured collagen or a Texas Red-labeled CNBr fragment CB7 from the alpha 1 chain of type I collagen. Kds obtained by fluid-phase fluorescence polarization binding assays increased upon oxidation about 17-fold for pFn and by 4-fold for the CGB domain. Comparison of CT-oxidized and native CGB domains by endogenous tryptophan fluorescence and CD spectra gave no indication of conformational changes. delta GH2O, the free energy of unfolding at infinite denaturant dilution, derived from guanidinium chloride denaturation curves, differed by less than 0.7 kcal/mol for the oxidized and native CGB domains, indicating essentially equivalent conformational stabilities. We show here that methionyl residues found at positions 412, 432, and 446 are the sites of the oxidative modification. Modification protection experiments carried out in the presence of gelatin demonstrated specific protection of otherwise oxidizable methionyl residues and preservation of high-affinity binding. These results implicate methionyl residues as functional in contributing to high-affinity binding interaction between fibronectin and gelatin.

Amino Acid Sequence↗
PubMed1993

Dialytic therapy for diabetic patients with terminal renal failure.

Diabetes mellitus is the leading cause of irreversible renal failure in the United States, Japan, and industrialized Europe. Of those diabetic patients begun on uremia therapy, 80% are treated with hemodialysis and 12% with peritoneal dialysis. Continuous ambulatory peritoneal dialysis is proposed by some as appropriate primary therapy for many diabetics with end-stage renal disease (ESRD), but peritonitis is common at one episode per 11 to 21 patient-months. Cardiovascular disease is accelerated in patients with diabetic ESRD and is the most common cause of death in diabetic dialysis patients. Although survival of diabetic ESRD patients is lower than that of nondiabetic patients (1- and 5-year patient survival is 74% and 19%, respectively, in diabetics and 78% and 40%, respectively, in all ESRD patients), morbidity and mortality figures have improved steadily over the past two decades. When possible, renal transplantation is advised as the treatment most likely to effect rehabilitation and long survival.

Cardiovascular Diseases↗
PubMed1993

The influence of donor age on function of renal allografts from live related donors.

To assess the influence of donor age on renal allograft outcome, we retrospectively analyzed all 169 consecutive cyclosporine-treated live related donor kidney transplants, of whom 40 were HLA identical siblings. All recipients were similar with respect to demographic and immunologic characteristics. Incidence of rejection episodes and graft survival rates at 1 and 5-year posttransplant were independent of donor age. Best renal function, as assessed by the mean of the lowest 3 serum creatinine concentration levels in the first 2 months posttransplant correlated positively with donor age, particularly among HLA mismatched male recipients (r = 0.4, P < 0.002). Short and intermediate term renal function was inferior, but stable in the older donor recipient group when compared to the younger cohort. Mean serum creatinine levels at 5 years in recipients of kidneys from older donors (age > 55 years) was 2.6 mg/dl compared to 1.7 and 1.9 mg/dl in recipients of kidneys from donors between the ages of 18-39 and 40-54 years, respectively (P < 0.001). In view of the universal shortage of organs and the negligible morbidity to the donors, our results should not discourage the use of kidneys from elderly (age > 55 years) donors.

Adult↗